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J Thorac Cardiovasc Surg 2003;126:1449-1454
© 2003 The American Association for Thoracic Surgery

Cardiopulmonary support and physiology

Cartilaginous metaplasia and calcification in aortic allograft is associated with transforming growth factor ß1 expression

P. Mathieu, MDa,*, J. C. Roussel, MDb, F. Dagenais, MDa, I. Anegon, MDb

a Quebec Heart Institute, Hôpital Laval, Sainte-Foy, Quebec, Canada
b Institut National de la Santé et de la Recherche Médicale (INSERM), Nantes, France

Received for publication January 27, 2002; revisions received April 30, 2002; accepted for publication September 17, 2002.

* Address for reprints: Patrick Mathieu, MD, Quebec Heart Institute, Hôpital Laval, 2725 Chemin Sainte-Foy, Sainte-Foy, Quebec GIV 4G5, Canada
prmathieu{at}hotmail.com


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
BACKGROUND: Calcification of homografts and vascular conduits is poorly understood. Mechanisms leading to calcification were studied in a rat model of aortic allografts.

METHODS: Rat aortas from Lew1W (RT1u) were transplanted into Lew1A (RT1a). Animals were killed at 30 days and 180 days, and aortic grafts were removed and analyzed for histologic and immunohistologic studies.

RESULTS: Intimal surface increased progressively over 6 months and was the site of important modifications. Intimal cellular population changed from a leukocyte (CD45, OX1-OX30)- and macrophage (CD68, ED-1)-based population at 30 days to predominantly {alpha}-smooth muscle actin-expressing cells at 180 days. At 180 days, allografts were characterized by an abundant extracellular matrix composed of collagen and elastic fibers associated with extensive calcification (von Kossa staining) located in the intima and media. Osteoblastic activity was present in calcified lesion as shown by alkaline phosphatase activity. At 180 days, numerous chondrocytes (protein S100-positive and {alpha}-smooth muscle actin-negative) were present focally in the media. However, double immunostaining revealed that a cellular population within the media with a chondrocyte-like morphology was {alpha}-smooth muscle actin-positive and S100-negative. Active form of transforming growth factor ß1 was expressed from 30 to 80 days in the medial and intimal layers.

CONCLUSIONS: These observations suggest that {alpha}-smooth muscle actin-positive cells within aortic allografts are eventually transformed to a chondrocyte-like structure, leading to vascular cartilaginous metaplasia associated with the expression of transforming growth factor ß1 and could be a potential pathway leading to extensive vascular wall calcification in allografts through endochondral ossification.

Calcification of vascular structures has been linked to different diseases such as atherosclerosis, cardiac valve disease, calcific uremic arteriolopathy, and transplant arteriosclerosis.1,2 Intimal calcification has been linked to end-stage chronic renal allograft rejection.3 Allograft conduit wall calcification is an important pathway leading to conduit graft failure, particularly in young patients.4 Mechanisms leading to allograft calcification are still ill defined. Until recently, vascular calcification and aortic allograft calcification were considered to be passive processes. However, observations suggest that calcified tissue express bone matrix proteins involved in the calcifying process.5 Elaboration of promineralizing matrix by the aortic allograft might be implicated in the heterotopic calcification process. The origin of promineralizing cells within the vascular wall remains unknown. It is hypothesized that these cells are either derived from blood-borne cells that invade the arterial wall or are native vascular cells with a dedifferentiation potential toward bone-forming cells.6

Cytokines or growth factors might be implicated in transformation of native or infiltrating vascular cells to calcifying vascular cells. In vitro, implication of growth factors such as transforming growth factor ß1 (TGF-ß-1) leading to transformation of vascular cells to calcifying cells has been described.7 We have used a model of aortic transplantation in the rat to study allograft arteriosclerosis and calcifying degeneration. Early expression of active form of TGF-ß1 after transplantation was associated with the development of graft cartilaginous metaplasia and endochondral ossification. Our data confirm that a population of {alpha}-smooth muscle actin-expressing cells within the aortic allograft are morphologically a chondrocyte-like structure that might represent an intermediary form preceding a fully mature chondrocyte.


   Methods
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 Methods
Results
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 References
 
Animals and aorta transplantation
Transplantations were performed using 250-g inbred male Lewis 1W (LEW.1W, haplotype RT1u) as donors and Lew.1A (RT1a) as recipients (CERJ, Le Genest St Isle, France). These animals are completely mismatched for the entire major histocompatibility complex region. The descending thoracic aorta was harvested from the donor and then anastomosed to the recipient's abdominal aorta below the renal arteries and above the aortic bifurcation. Anastomoses were done as previously described8 using termino-lateral technique at both proximal and distal ends of the allograft, and the native aorta was ligated between the anastomosis. Grafted aortas (allografts and isografts) were harvested at 30 (n = 6) and 180 (n = 4) days after transplantation. Harvested segments were cut into 2 equal pieces, and 1 segment was fixed with formaldehyde 10% for histologic processing. The proximal and distal anastomosis parts were discarded to eliminate potential healing effects. Cross sections of fixed segments were stained with hematoxylin-eosin (H&E), von Weigert (for elastic fibers), trichrome Masson (for collagen), and von Kossa (for mineralization). The other segment was embedded in optimum cutting temperature (OCT) compound (TissueTek, Miles Laboratories, Elkhart, Ind) and frozen liquid nitrogen for immunohistologic analysis.

Immunohistologic analysis
Immunohistology was performed in cryostat sections as previously described in detail.9 Immunohistologic analysis of infiltrating leukocytes was performed using the following mouse Mab: a mixture of 2 anti-leukocytes CD45 Mabs (OX1 and OX30), anti-monocyte/macrophage CD68 (ED1), anti-{alpha}ß T-cell receptor (TCR) (R.7.3) (all from ECACC, Wiltshire, United Kingdom), anti-{alpha} smooth muscle actin (SMC) (Sigma, St Louis, Mo), anti-active form of TGF-ß1 (R&D Systems, Inc, Minneapolis, Minn), and an irrelevant mouse Mab (3G8, anti-human CD16). Slides were then incubated with a biotin-conjugated anti-mouse immunoglobulin antibody (Vector Laboratories, Burlingame, Calif), followed by horseradish peroxide-conjugated streptavidin (Vector Laboratories). S100 protein (a useful marker for chondrocytes) was detected using rabbit Mab (DAKO Corporation, Carpinteria, Calif). Biotin-conjugated anti-rabbit antibody were from Jackson Laboratory, Bar Harbor, Maine. Binding of these antibodies was detected by incubation with horseradish peroxidase-conjugated streptavidin and VIP or DAB substrate (Vector Laboratories). Double immunostaining for S100 and {alpha} SMC actin was used with the following protocol: slides were first incubated with mouse anti-{alpha} SMC actin (Sigma) and followed with biotin-conjugated anti-mouse antibody (Jackson Laboratories). Binding of biotin-conjugated antibody was detected by incubation with horseradish peroxidase-conjugated straptavidin and DAB substrate (Vector Laboratories). Then S100 protein was detected using rabbit anti-S100 protein (DAKO) followed by phosphatase alkaline-conjugated antibody and revealed with Vector Blue (Vector Laboratories) and levamisol.

Alkaline phosphatase activity
Cryostat sections of aortic graft tissue were incubated for an hour with Vector Blue alkaline phosphatase substrate kit (Vector Laboratories). Then slides were mounted for analysis.


   Results
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Discussion
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At day 30, allografts were heavily infiltrated by leukocytes (CD45), and the cellular population was composed of T cells ({alpha}ß TCR) and macrophages (ED1) infiltrating intima and adventitia (Figure 1). At day 180, intima of allografts showed an impressive decrease of inflammatory cellular population such as leukocytes (CD45), macrophages (ED1), and T cells ({alpha}ß TCR; Figure 1). At day 180 cellular population within the intima was predominantly represented with {alpha}-smooth muscle actin-expressing cells (data not shown). At day 180, cells with the histologic appearance of chondrocytes were present focally in the intima of all (4/4 animals) allografts (Figure 2). Immunohistochemical studies revealed that most of these cells were S100-positive and therefore were chondrocytes (Figure 2). A few cells had a chondrocyte-like structure but were S100-negative. Double immunostaining technique showed that these chondrocyte-like cells were S100-negative and {alpha}-smooth muscle actin-positive, suggesting that these cells might be derived from smooth muscle cell precursor or other {alpha}-smooth muscle actin-expressing cells such as pericyte (Figure 3).



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  		Figure 1. Cellular infiltration of aortic allografts at 1 month and 6 months. Immunohistology for CD45 (leukocytes), CD68 (macrophages), and T cells ({alpha}ß-TCR).

 


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  		Figure 2. A, Aortic allografts at 6 months. H&E staining shows cells with histologic appearance of chondrocytes. B, Immunohistologic preparation showing S100-positive cells within the intima confirming the presence of chondrocytes.

 


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  		Figure 3. Double immunostaining technique showed that a few cells within the intima of aortic allograft at 6 months had a chondrocyte-like structure and were {alpha}-actin-positive (brown cell) and S100-negative, whereas most of them were S100-positive (blue cell), indicating the presence of chondrocytes.

 
Because chondrogenesis is often followed by osteogenesis, we tested harvested allografts at day 180 for the presence of calcification. Staining for mineralized tissue (von Kossa) showed the presence of calcification located at the interface between the intima and the media with invasion of the latter layer, and these calcified lesions had alkaline phosphatase activity, indicating osteogenesis activity (Figure 4). Furthermore, calcified arteries were fibrotic, as demonstrated with collagen deposition (trichrome Masson staining) throughout the vascular wall (Figure 4). Elastic fibers (von Weigert staining) were detected in the intima following a reticular pattern (Figure 4).



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  		Figure 4. A, Aortic allografts at 6 months showed extensive calcification located between the intima and media (von Kossa staining). B, Osteoblastic activity was confirmed with alkaline phosphatase staining (blue). C, Presence of extensive fibrosis was confirmed with trichrome Masson staining. D, Deposition of a reticular pattern of elastin was found at the basal layer of the intima.

 
To investigate whether TGF-ß1 might be associated with vascular cartilaginous metaplasia, we conducted immunohistologic studies on aortic grafts. In control isografts, TGF-ß1 was expressed in the media at both 30 and 180 days. However, at 30 days, allografts had a stronger expression of TGF-ß1 located in the intima and the media that persisted until day 180. At day 180, TGF-ß1 was expressed by intimal cells, medial cells, and by chondrocyte or chondrocyte-like cells (Figure 5).



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  		Figure 5. At 6 months, immunohistochemical studies showed the expression of the active form of TGF-ß1 in aortic isografts (A), which was located in the media. In allografts (B), a stronger expression of TGF ß1 was found in the media and intima.

 

   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
Clinically, allograft conduits used for complex congenital heart disease or aortic valve allograft are prone to calcification, particularly in young patients. The calcification process in either aortic conduit or aortic valve is associated with functional failure of the graft. However, allograft calcification and failure proceeds through still ill-defined mechanisms. This experimental study showed that cartilaginous metaplasia and osteogenesis was associated with heterotopic calcification in aortic allograft. These calcifications were specific to aortic allografts and were not detected in either isograft or native arteries (data not shown) of the transplanted animals. Therefore, host immune responses against alloantigens are involved in this model of heterotopic calcification. We explored in vivo mechanisms leading to heterotopic calcification, and we found that a population of precursor cells in the arterial wall expressing {alpha}-smooth muscle actin might be involved in the metaplasic process and subsequent calcification.

Heterotopic calcification is an active process that involves osteoblastic activity.10 Endochondral osteogenesis similar to that observed in the growth plate has been shown to be present in human stenotic heart valves.10 Secretion of promineralizing extracellular matrix composed of different proteins such as osteocalcin, osteopontin, and bone morphogenetic protein-2 (BMP-2) have been documented in calcified atherosclerotic lesions, indicating a process similar to what is observed in bone mineralization.11-13 Alkaline phosphatase activity has been demonstrated in our calcified allografts, suggesting similarities to osteogenesis. In vitro, alkaline phosphatase activity in bovine smooth muscle cells has been shown to be essential for the mineralization process.14 Therefore, our model suggests that a process similar to osteogenesis is involved in aortic allograft calcification. This process of calcification was triggered by the immune response since isografts retained a normal histology. This is in accordance with human studies that suggest an immune response against alloantigens in homografts recipients.15,16 However, others studies have stressed the role of preservation, donor age, and the viability of homografts as factors involved in allografts structural failure.17

Mineralization of vascular tissue proceeds from differentiation of pluripotent mesenchymal cells to calcifying cells. In vitro, smooth muscle cells exposed to vitamin D, warfarin, and high phosphate concentration induced mineralization process, with development of osteoblastic differentiation markers.18,19 In vitro, a population of bovine and human cells from the arterial wall undergoes spontaneous calcification.6 These calcifying vascular cells share many similarities with microvascular pericyte. Pericytes have been reported to be precursors of osteoblasts and smooth muscle cells.20 Heterotopic endochondral ossification in atherosclerotic plaque has been shown to proceed from cells that resemble microvascular pericytes.21 We found in aortic allografts that mature chondrocytes expressing S100 protein were present in the vascular wall. However, a small population of chondrocyte-like cells were S100-negative but {alpha}-smooth muscle actin-positive, suggesting that a precursor cell expressing {alpha}-smooth muscle actin might be involved in the metaplasic process. Both smooth muscle cells and pericytes have been shown to express {alpha}-smooth muscle actin. Therefore, 1 or both of these cells might be involved in the pathologic process of heterotopic calcification through endochondral ossification.

Heterotopic calcification through endochondral ossification from pericytes has been shown to be influenced by cytokines and growth factors produced by immune cells.22 Members of the TGF-ß family have been associated with chondrogenesis in the skeletal system.23 Cartilaginous metaplasia in the cardiovascular system has been described in human heart valves and in aortas of atherosclerotic mice but no association with TGF-ß was made.24,25 However, in vivo, transfection and local expression of TGF-ß1 in rat aorta induced a cartilaginous metaplasia that was reversible when TGF-ß1 expression was stopped.26 In vitro, cellular populations within the aortic wall that spontaneously calcify, called "calcifying vascular cells," are stimulated by TGF-ß17 These calcifying vascular cells are {alpha}-smooth muscle actin-positive, suggesting that they are derived from either smooth muscle cells or from pericytes. In aortic allografts, we found that cartilaginous metaplasia and calcification was associated with early expression of TGF-ß1 throughout the study period until 6 months after grafting. However, in our model no causal effect can be drawn from the association of TGF-ß1 expression and vascular cartilaginous metaplasia as it is an observational study. In fact, the pathogenic role of the pleiotropic growth factor, TGF-ß1, is still subjected to controversies and to some point poorly understood.27 Several studies have associated TGF-ß1 expression with the development of vascular disease, whereas others studies have demonstrated that the expression of TGF-ß1 prevents arterial vascular disease.27-29 Apparently such diverse effect of TGF-ß1 on the biology of the vascular system might be related to the signals delivered according to the state of differentiation of the cells and their qualitative and quantitative expression of different type of TGF receptors.30

Interactions between different inflammatory pathways and specific mechanisms leading to heterotopic calcification are still poorly understood. Different molecules such as vitamin D, phosphate, steroids, warfarin, and cytokines might be involved in the calcification process.14,31,32 It is likely that activation of graft infiltrating cells and native vascular cells leads to local production of cytokines and growth factors that are involved in the heterotopic process of calcification of the aortic allograft. However, the specific nature and interaction between different cytokines and growth factors leading to transformation of cells into bone-forming cells is still an embryonic field. Prevention of heterotopic calcification of the aortic allograft would lead to better long-term function of aortic allografts. Therefore, understanding expression and physiology of growth factors in the aortic allograft could provide important insight into the pathophysiology of heterotopic calcification process.


   References
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 Discussion
 References
 

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